WO2024227231A1 - Protéines de fusion à base de nampt - Google Patents

Protéines de fusion à base de nampt Download PDF

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Publication number
WO2024227231A1
WO2024227231A1 PCT/AU2024/050432 AU2024050432W WO2024227231A1 WO 2024227231 A1 WO2024227231 A1 WO 2024227231A1 AU 2024050432 W AU2024050432 W AU 2024050432W WO 2024227231 A1 WO2024227231 A1 WO 2024227231A1
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Prior art keywords
fusion protein
amino acid
acid sequence
cell
polypeptide
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Peter Currie
Mikaël MARTINO
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Monash University
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Monash University
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Priority claimed from AU2023901320A external-priority patent/AU2023901320A0/en
Application filed by Monash University filed Critical Monash University
Priority to EP24799791.9A priority Critical patent/EP4705440A1/fr
Priority to KR1020257039208A priority patent/KR20260010708A/ko
Priority to AU2024264943A priority patent/AU2024264943A1/en
Publication of WO2024227231A1 publication Critical patent/WO2024227231A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1077—Pentosyltransferases (2.4.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/06—Anabolic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/02—Pentosyltransferases (2.4.2)
    • C12Y204/02012—Nicotinamide phosphoribosyltransferase (2.4.2.12), i.e. visfatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor

Definitions

  • the present invention relates to productive tissue repair and regeneration, and in particular fusion proteins, compositions including said fusion proteins, and methods of using said fusion proteins or compositions for productive tissue repair and regeneration.
  • Skeletal muscle typically forms approximately 40% of a body mass in human adults. It is formed during development by myogenesis wherein paired blocks of paraxial mesoderm known as somites give rise to a transitory myotome that forms muscle stem cells and expands to form an integrated and complex musculature through fusion of myoblasts to the surface of myotubes. In a further stage of myogenesis, muscle stem cells (called satellite cells) migrate to occupy a niche between the sarcolemma and basal lamina of individual myofibers. Amniotes are born with a full set of muscle fibres and, in adults, muscle repair is generally effected through an increase in the size of existing fibres.
  • the satellite cell is archetypal of a unipotent tissue-resident stem cell that occupies a specific anatomical niche within a differentiated tissue.
  • Decades of research have revealed the extraordinary capacity of this system to effectively coordinate muscle repair in response to a wide variety of insults.
  • transplantation of isolated muscle stem cells has yet to provide therapeutic impact, and pro-regenerative treatments that stimulate muscle stem cells are entirely lacking at this juncture.
  • a fusion protein comprising:
  • the Fc region has a reduced affinity for an Fc receptor compared to wild-type or naturally occurring Fc regions.
  • NAMPT cif polypeptide and Fc region of an antibody may be joined directly or via a linker sequence.
  • the NAMPT cif polypeptide is C-terminal to the Fc region of the antibody.
  • the N-terminus of the NAMPT cif polypeptide is joined directly or via a linker sequence to the C-terminus of the Fc region of an antibody.
  • the NAMPT cif polypeptide is N-terminal to the Fc region of the antibody.
  • the C-terminus of the NAMPT cif polypeptide is joined directly or via a linker sequence to the N-terminus of the Fc region of an antibody.
  • the fusion protein comprises a first NAMPT cif polypeptide that is N-terminal to the Fc region of the antibody and a second NAMPT cif polypeptide that is C-terminal to the Fc region of the antibody.
  • the C-terminus of the first NAMPT cif polypeptide is joined directly or via a linker sequence to the N-terminus of the Fc region of an antibody.
  • the N-terminus of the second NAMPT cif polypeptide is joined directly or via a linker sequence to the C-terminus of the Fc region of an antibody.
  • the NAMPT cytokine finger (cif) polypeptide comprises, consists essentially of or consists of an amino acid sequence of full length NAMPT cif.
  • the NAMPT cytokine finger (cif) polypeptide comprises, consists essentially of or consists of an amino acid sequence of a truncated NAMPT cif.
  • the NAMPT cytokine finger (cif) polypeptide comprises, consists essentially of or consists of an amino acid sequence of a fragment of NAMPT cif.
  • the NAMPT cytokine finger (cif) polypeptide comprises, consists essentially of or consists of an amino acid sequence of NAMPT402-491.
  • the numbering corresponds to that of full length NAMPT as shown in SEQ ID NO: 19.
  • the NAMPT cytokine finger (cif) polypeptide comprises, consists essentially of or consists of an amino acid sequence of NAMPT414-491.
  • the numbering corresponds to that of full length NAMPT as shown in SEQ ID NO: 19.
  • the NAMPT cytokine finger (cif) polypeptide comprises, consists essentially of or consists of an amino acid sequence of NAMPT422-491.
  • the numbering corresponds to that of full length NAMPT as shown in SEQ ID NO: 19.
  • the NAMPT cytokine finger (cif) polypeptide comprises, consists essentially of or consists of an amino acid sequence of NAMPT430-491.
  • the numbering corresponds to that of full length NAMPT as shown in SEQ ID NO: 19.
  • the NAMPT cytokine finger (cif) polypeptide comprises, consists essentially of or consists of an amino acid sequence of NAMPT436-491.
  • the numbering corresponds to that of full length NAMPT as shown in SEQ ID NO: 19.
  • the fusion protein may further comprise a signal sequence, for example an IL-2 signal sequence.
  • the signal sequence comprises, consists essentially of or consists of the amino acid sequence as shown in SEQ ID NO: 36.
  • the NAMPT cif polypeptide comprises, consists essentially of or consists of the amino acid sequence set out in any one of SEQ ID Nos:
  • the NAMPT cif polypeptide comprises, consists essentially of or consists of a truncated amino acid sequence of SEQ ID NO: 1.
  • the truncated NAMPT cif may have an N-terminal truncation and/or a C-terminal truncation compared to full length NAMPT cif.
  • the N- and/or C-terminal truncation is at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10,
  • the truncation is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or
  • the truncation is of the N-terminal residues 1-12, 1-20, 1- 28 or 1-35 of cif.
  • the truncation is of, or equivalent to, the N-terminal residues 1-12, 1-20, 1-28 or 1-35 of the amino acid sequence set forth in SEQ ID NO: 1.
  • the NAMPT cif polypeptide comprises, consists essentially of or consists of an amino acid sequence of any one of SEQ ID Nos: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 , or an amino acid sequence that is equal to, or at least, about
  • the NAMPT cif polypeptide comprises, consists essentially of or consists of an amino acid sequence of any one of SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 , or an amino acid sequence that is equal to, or at least, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence of any one of SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11.
  • % identity or identical to a sequence means that the polypeptide has the same length, for example number of amino acids, but the amino acids across that length are only 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.
  • the only differences in amino acid identity is a result of conservative substitutions (for example those outlined in Table 3 below).
  • the NAMPT cif polypeptide is equal to, or less than, about 110, about 109, about 108, about 107, about 106, about 105, about 104, about 103, about 102, about 101 , about 100, about 99, about 98, about 97, about 96, about 95, about 94, about 93, about 92, about 91 , about 90, about 89, about 88, about 87, about 86, about 85, about 84, about 83, about 82, about 81 , about 80, about 79, about 78, about 77, about 76, about 75, about 74, about 73, about 72, about 71 , about 70, about 69, about 68, about 67, about 66, about 65, about 64, about 63, about 62, about 61 , about 60, about 59, about 58, about 57, or about 56 amino acids in length.
  • the NAMPT cif polypeptide is equal to, or less than, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101 , 100, 99, 98, 97, 96, 95, 94, 93, 92, 91 , 90, 89, 88, 87, 86, 85, 84, 83, 82, 81 , 80, 79, 78, 77, 76, 75, 74, 73, 72, 71 , 70, 69, 68, 67, 66, 65, 64, 63, 62, 61 , 60, 59, 58, 57, or 56 amino acids in length.
  • the amino acid sequence of the NAMPT cif polypeptide that is equal to, or at least, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to an amino acid sequence of any one of SEQ ID Nos: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 is equal to, or less than, about 110, about 109, about 108, about 107, about 106, about 105, about 104, about 103, about 102, about 101 , about 100, about 99, about 98, about 97, about 96, about 95, about 94, about 93, about 92, about 91 , about 90, about 89, about 88, about 87, about 86, about 85, about 84, about 83, about 82, about
  • the amino acid sequence of the NAM PT cif polypeptide that is equal to, or at least, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence of any one of SEQ ID Nos: 1 , 2 ,3 ,4, 5, 6, 7, 8, 9, 10 or 11 is equal to, or less than, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101 , 100, 99, 98, 97, 96, 95, 94, 93, 92, 91 , 90, 89, 88, 87, 86, 85, 84, 83, 82, 81 , 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66
  • the NAMPT cif polypeptide comprises an amino acid sequence having 1 , 2, 3, 4, 5, 6, 7 or 8 conservative (for example those outlined in Table 3 below) or non-conservative amino acid substitutions, deletions or additions to the above sequences, and retains CCR5 or tissue stem cell interacting activity.
  • conservative or non-conservative amino acid substitutions, deletions or additions are not of the amino acids 431 to 435 or 472 to 491 (numbering corresponding to human NAMPT, e.g. SEQ ID NO: 19).
  • the NAMPT cif polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 8 with an N- terminal truncation.
  • the N-terminal truncation is less than 6 amino acids.
  • any polypeptide as described herein comprises the C- terminal alpha helix present in the cif motif.
  • the NAMPT cif polypeptide consists of an amino acid sequence having equal to, or at least, 85% sequence identity to the amino acid sequence of SEQ ID NO: 1.
  • the amino acid sequence of the polypeptide is equal to, or at least, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1.
  • the NAMPT cif polypeptide consists of an amino acid sequence having equal to, or at least, 85% sequence identity to the amino acid sequence of SEQ ID NO: 4 or 5.
  • the amino acid sequence of the polypeptide is equal to, or at least, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4 or 5.
  • the NAMPT cif polypeptide consists of an amino acid sequence having equal to, or at least, 85% sequence identity to the amino acid sequence of SEQ ID NO: 6 or 7.
  • the amino acid sequence of the polypeptide is equal to, or at least, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6 or 7.
  • the NAMPT cif polypeptide consists of an amino acid sequence having equal to, or at least, 85% sequence identity to the amino acid sequence of SEQ ID NO: 8 or 9.
  • the amino acid sequence of the polypeptide is equal to, or at least, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8 or 9.
  • the NAMPT cif polypeptide consists of an amino acid sequence having equal to, or at least, 85% sequence identity to the amino acid sequence of SEQ ID NO: 10 or 11.
  • the amino acid sequence of the polypeptide is equal to, or at least, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10 or 11.
  • the truncated NAMPT cif has an N terminal truncation of, or equivalent to, residues 402 to 413 of SEQ ID NO: 19, and wherein the polypeptide has at least 85% sequence identity to the amino acid sequence of 414 to 491 of SEQ ID NO: 19.
  • the amino acid sequence of the polypeptide is equal to, or at least, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of 414 to 491 of SEQ ID NO: 19.
  • the truncated NAMPT cif has an N terminal truncation of, or equivalent to, residues 402 to 421 of SEQ ID NO: 19, and wherein the polypeptide has at least 85% sequence identity to the amino acid sequence of 422 to 491 of SEQ ID NO: 19.
  • the amino acid sequence of the polypeptide is equal to, or at least, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of 422 to 491 of SEQ ID NO: 19.
  • the truncated NAM PT c if has an N terminal truncation of, or equivalent to, residues 402 to 429 of SEQ ID NO: 19, and wherein the polypeptide has at least 85% sequence identity to the amino acid sequence of 430 to 491 of SEQ ID NO: 19.
  • the amino acid sequence of the polypeptide is equal to, or at least, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of 430 to 491 of SEQ ID NO: 19.
  • the truncated NAM PT c if has an N terminal truncation of, or equivalent to, residues 402 to 435 of SEQ ID NO: 19, and wherein the polypeptide has at least 85% sequence identity to the amino acid sequence of 436 to 491 of SEQ ID NO: 19.
  • the amino acid sequence of the polypeptide is equal to, or at least, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of 436 to 491 of SEQ ID NO: 19.
  • the fusion protein may be in monomeric, dimeric or multimeric form.
  • any polypeptide described herein may be modified to allow homo or heterodimerisation.
  • the modification may be the addition of an amino acid, either natural or non-natural, that forms a covalent bond, for example as cysteine that forms a disulphide bond.
  • the addition of an amino acid, such as a cysteine, that forms a covalent bond is at the N- or C-terminus of the polypeptide.
  • the polypeptide comprises, consists essentially of or consists of an amino acid sequence of any one of SEQ ID Nos: 1, 3, 4, 5, 6, 7, 8, 9, 10 or 11 with an additional cysteine at the N- or C-terminus.
  • a dimeric fusion protein formed by covalent bonded, preferably disulphide bonded, monomers of a polypeptide described herein.
  • the fusion protein comprises, consists essentially of or consists of 2 or more full length NAM PT cif molecules, truncated NAM PT cif molecules, or fragments thereof as described herein.
  • the Fc region of an antibody may be an Fc region of an IgG, IgA, IgD, IgE, or IgM.
  • the Fc region is from an IgG antibody, such as an lgG1 , an lgG2, an lgG2b, an lgG3 or an lgG4 antibody.
  • the Fc region of the fusion protein comprises two heavy chain fragments, more preferably the CH2 and CH3 domains of said heavy chain.
  • the Fc region of the fusion protein comprises a hinge region.
  • the hinge region may be derived from an immunoglobulin-like molecule.
  • the hinge region may comprise or consist of a hinge region from an lgG1 , lgG2, lgG3 or lgG4, including a fragment thereof.
  • the Fc region of an antibody may not comprise one or more of a VH, VL, light chain CH1 and heavy chain CH1.
  • the Fc region of an antibody comprises, consists essentially of or consists of a hinge region, CH2 and CH3 domain.
  • the fusion protein may comprise a linker between the NAM PT cif polypeptide and the Fc region of the antibody, such as between the C- terminus of the Fc region and the N-terminus of the NAMPT cif polypeptide.
  • the Fc region preferably comprises one or more amino acid substitutions for reducing affinity for the Fc receptor (FcR, including any of FcyRI, FcyRII and FcyRIII) and thereby reducing the ability of the fusion protein to elicit antibody-dependent cell- mediated toxicity (ADCC).
  • FcR Fc receptor
  • Such substitutions are well known in the art and include, but are not limited to the “DANA”, and “LALA” amino acid substitutions and variations thereof, as further defined herein.
  • the fusion protein has an affinity for FcR that is less than about 250 nM, preferably less than 500 nM, less than 1000 nM, most preferably less than 2000 nM.
  • the Fc region of an antibody may include one or more amino acid substitutions for increasing the serum half-life of the Fc and/or the fusion protein.
  • the amino acid substitutions for increasing the serum halflife of the Fc and/or the fusion protein is a substitution at one or more of M252, S254 and T256.
  • the substitutions are one or more of M252Y, S254T and T256E.
  • the Fc region of an antibody comprises, consists essentially of or consists of an amino acid sequence as shown in SEQ ID NOs: 38 or 40.
  • the fusion protein may comprise a linker between the NAM PT cif polypeptide and the Fc region of the antibody, such as between the N- terminus of the Fc regions and the C-terminus of the NAM PT polypeptide.
  • the linker may have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acids.
  • the linker is a peptide linker.
  • the peptide linker links a NAMPT polypeptide with an Fc region of an antibody.
  • the peptide linker can include the amino acid sequence Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS; SEQ ID NO: 72) or Gly-Gly-Gly-Gly-Ser (GGGGS; SEQ ID NO: 73).
  • the peptide linker can include the amino acid sequence GGGGS (a linker of 6 amino acids in length) or even longer.
  • the linker may a series of repeating glycine and serine residues (GS) of different lengths, i.e.
  • n is any number from 1 to 15 or more.
  • the linker may be (GS)3 (i.e., GSGSGS; SEQ ID NO: 74) or longer (GS)11 or longer.
  • n can be any number including 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more. Fusion proteins having linkers of such length are included within the scope of the present invention.
  • n is no more than 3 (ie such that when n equals 3 the linker is GSGSGS; SEQ ID NO: 74).
  • the linker comprises or consists of the sequence shown in SEQ ID NO: 41.
  • the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 42 to 61.
  • a fusion protein as described herein stimulates muscle stem cell (e.g. satellite) proliferation to a level that is equal to or greater than full length NAMPT, for example comprising, consisting essentially of or consisting of SEQ ID NO: 19, or equal to or greater than full length NAMPTcif without an Fc region of an antibody, for example comprising, consisting essentially of or consisting of SEQ ID NO: 1 or 2.
  • muscle stem cell (e.g. satellite) proliferation is determined by an assay described herein, including in Example 1.
  • a fusion protein described herein stimulates TLR4 activation to a significantly lower level than full length NAM PT.
  • a polypeptide or fusion protein stimulates TLR4 activation to the same, or not-significantly different, level as the cytokine finger motif of NAMPT without an Fc region of an antibody (for example, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1).
  • the fusion protein is a dimer wherein each monomer comprises:
  • each monomer in the dimer comprises, consists essentially of or consists of a hinge region, CH2, CH3 and NAMPT cif polypeptide.
  • each monomer has the following arrangement, from N-terminus to C-terminus: a hinge region, CH2, CH3 and NAMPT cif polypeptide.
  • the monomer comprises, consists essentially of or consist of an amino acid sequence as shown in any one of SEQ ID NOs: 42 to 61.
  • the present invention also provides isolated nucleic acids encoding a fusion protein as described herein, for example, comprising, consisting essentially of or consisting of the amino acid sequence of any one of SEQ ID Nos: 42 to 61.
  • a nucleic acid molecule encoding a fusion protein described herein comprises, consists essentially of or consists of the polynucleotide sequence set out in any one of SEQ ID NO: 68 to 71, or a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.
  • the nucleic acid molecule may be an RNA or DNA or RNA : DNA or a chemically modified form thereof.
  • the nucleic acid may be in the form of a viral or non-viral vector.
  • the present invention provides vectors comprising said nucleic acids, optionally, operably linked to control sequences.
  • the present invention provides host cells containing the vectors, and methods for producing and optionally recovering the fusion proteins.
  • the present invention provides a cell expressing a fusion protein as described herein.
  • the present invention provides compositions providing chemokine receptor interaction or binding activity or muscle tissue stem cell interacting activity for use in stimulating muscle regeneration.
  • the present application provides compositions providing chemokine receptor interaction or binding activity or satellite cell binding or interacting activity for use in stimulating muscle regeneration without fibrosis or substantially without fibrosis.
  • the chemokine receptor is a CCR5 chemokine receptor or a tissue stem cell receptor that binds NAMPT, including a tissue stem cell receptor that binds NAMPTcif.
  • the composition comprising a cell or other agent that provides CCR5 interacting activity, binds to tissue stem cells, particularly muscle stem cells.
  • the composition comprises a fusion protein, nucleic acid, vector, or cell as described herein and a pharmaceutically acceptable carrier, diluent or excipient.
  • the composition may further comprise one, two or all of
  • tissue stem cell such as a satellite cell
  • precursor therefore or progeny thereof
  • promoting muscle stem cell chemokine receptor signalling is particularly useful in treating subjects with a muscle injury including volumetric muscle loss injuries or muscle degeneration/atrophy, or muscular or neuromuscular impairments, muscular or neuromuscular degenerative conditions, myopathy, or the propensity therefore.
  • chemokine receptor binding activity is provided in the form of a cell such as a macrophage or stem cell expressing a fusion protein as described herein.
  • the chemokine receptor is a CCR5 receptor.
  • the CCR5 receptor is a tissue stem cell or tissue stem cell progeny CCR5 receptor.
  • the CCR5 receptor is a satellite cell or satellite cell progeny CCR5 receptor.
  • in vitro, in vivo and ex vivo applications are contemplated.
  • the present invention provides a method of stimulating proliferation of a stem cell, such as satellite cell proliferation, the method comprising administering to a cell or subject an effective amount of a fusion protein, nucleic acid, vector, composition or cell as described herein, thereby stimulating proliferation of a stem cell.
  • the present invention provides use of a fusion protein, nucleic acid, vector, composition or cell as described herein as described herein in the manufacture of a medicament for stimulating proliferation of a stem cell, such as satellite cell proliferation.
  • the present invention provides a fusion protein, nucleic acid, vector, composition or cell as described herein as described herein for use in stimulating proliferation of a stem cell, comprising administering to a cell or subject an effective amount of the fusion protein, nucleic acid, vector, composition or cell, thereby stimulating proliferation of a stem cell.
  • the present invention provides a method of stimulating muscle tissue regeneration in a subject, the method comprising administering to a muscle of the subject an effective amount of a fusion protein, nucleic acid, vector, composition or cell as described herein, thereby stimulating muscle tissue regeneration.
  • the present invention provides use of a fusion protein, nucleic acid, vector, composition or cell as described herein as described herein in the manufacture of a medicament for stimulating muscle tissue regeneration in a subject.
  • the present invention provides a fusion protein, nucleic acid, vector, composition or cell as described herein as described herein for use in stimulating muscle tissue regeneration in a subject, comprising administering to the subject an effective amount of the fusion protein, nucleic acid, vector, composition or cell, thereby stimulating muscle tissue regeneration.
  • the fusion protein is a CCR5 agonist. That is, it stimulates receptor signalling or downstream events such as satellite cell activation and proliferation.
  • the fusion protein specifically activates tissue stem cells.
  • the fusion protein specifically activates satellite cells.
  • tissue regeneration stimulated by the method is associated with minimal fibrosis.
  • the present application provides fusion proteins, compositions, cells and methods for reducing fibrosis development in a patient or biological tissue subject to regenerative treatment.
  • the present application provides a method suitable for regenerating muscle tissue in vitro, in vivo or ex vivo. Accordingly, fusion protein, nucleic acid, vector, composition or cell described herein is proposed for use, or for use in, stem cell based therapies and tissue engineering, including for use in the manufacture of medicaments thereto. In another embodiment fusion protein, nucleic acid, vector, composition or cell described herein is for use, or for use in, artificial meat production in vitro.
  • the present invention provides a method of stimulating muscle tissue regeneration, the method comprising administering to a muscle an effective amount of a fusion protein, nucleic acid, vector, composition or cell as described herein, wherein the fusion protein, nucleic acid, vector, composition or cell as described herein binds to satellite cells and stimulates satellite cell activation, myoblast proliferation and muscle regeneration and the absence of substantial fibrosis (scar formation).
  • the present invention provides use of a fusion protein, nucleic acid, vector, composition or cell as described herein as described herein in the manufacture of a medicament for stimulating muscle tissue regeneration, wherein the fusion protein, nucleic acid, vector, composition or cell as described herein binds to satellite cells and stimulates satellite cell activation, myoblast proliferation and muscle regeneration and the absence of substantial fibrosis (scar formation).
  • the present invention provides a fusion protein, nucleic acid, vector, composition or cell as described herein as described herein for use in stimulating muscle tissue regeneration, comprising administering to the cell or subject an effective amount of the fusion protein, nucleic acid, vector, composition or cell, thereby stimulating muscle tissue regeneration, wherein the fusion protein, nucleic acid, vector, composition or cell binds to satellite cells and stimulates satellite cell activation, myoblast proliferation and muscle regeneration and the absence of substantial fibrosis (scar formation).
  • the present invention provides a method of stimulating muscle tissue regeneration in a subject where inflammation is undesirable, the method comprising administering to a muscle an effective amount of a fusion protein, nucleic acid, vector, composition or cell as described herein, thereby stimulating muscle tissue regeneration in this subject.
  • the present invention provides use of a fusion protein, nucleic acid, vector, composition or cell as described herein in the manufacture of a medicament for stimulating muscle tissue regeneration in a subject where inflammation is undesirable.
  • the present invention provides a fusion protein, nucleic acid, vector, composition or cell as described herein for use in stimulating muscle tissue regeneration in a subject where inflammation is undesirable, comprising administering to a muscle an effective amount of the fusion protein, nucleic acid, vector, composition or cell as described herein, thereby stimulating muscle tissue regeneration in this subject.
  • the inflammation that is undesirable is inflammation mediated by TLR activation, preferably TLR4 activation.
  • the subject may be diagnosed with an inflammatory myopathy.
  • inflammatory myopathies include polymyositis, dermatomyositis, inclusion body myositis, necrotizing autoimmune myopathy.
  • the present invention provides a method of treating an inflammatory myopathy in a subject, the method comprising administering to a muscle of the subject an effective amount of a fusion protein, nucleic acid, vector, composition or cell as described herein, thereby treating an inflammatory myopathy.
  • the inflammatory myopathy is polymyositis, dermatomyositis, inclusion body myositis or necrotizing autoimmune myopathy.
  • the present invention provides use of a fusion protein, nucleic acid, vector, composition or cell as described herein in the manufacture of a medicament for treating an inflammatory myopathy in a subject.
  • the inflammatory myopathy is polymyositis, dermatomyositis, inclusion body myositis or necrotizing autoimmune myopathy.
  • the present invention provides a fusion protein, nucleic acid, vector, composition or cell as described herein for use in treating an inflammatory myopathy in a subject, comprising administering to a muscle of the subject an effective amount of the fusion protein, composition or cell as described herein, thereby treating an inflammatory myopathy.
  • the inflammatory myopathy is polymyositis, dermatomyositis, inclusion body myositis or necrotizing autoimmune myopathy.
  • Reference to NAMPT and CCR5 includes homologues and orthologs thereof include from any animal including mammals, non-mammalian vertebrates, fish and birds.
  • the present application provides a method of stimulating muscle tissue regeneration, the method comprising administering to a muscle an effective amount of a composition comprising a cell comprising or encoding a fusion protein as described herein, and optionally a component that enhances delivery to or retention in the muscle, wherein the fusion protein as described herein binds to satellite cells and stimulates myoblast proliferation and muscle regeneration.
  • the present application provides use of a composition comprising a cell comprising or encoding a fusion protein as described herein, and optionally a component that enhances delivery to or retention in the muscle, in the manufacture of medicament for stimulating muscle tissue regeneration, wherein the fusion protein as described herein binds to satellite cells and stimulates myoblast proliferation and muscle regeneration.
  • the present application provides a composition comprising a cell comprising or encoding a fusion protein as described herein, and optionally a component that enhances delivery to or retention in the muscle for use in stimulating muscle tissue regeneration, comprising administering to a muscle an effective amount of a composition comprising a cell comprising or encoding a fusion protein as described herein, and optionally a component that enhances delivery to or retention in the muscle, wherein the fusion protein as described herein binds to satellite cells and stimulates myoblast proliferation and muscle regeneration.
  • the cell is a macrophage.
  • the macrophage is isolated from tissue.
  • the macrophage is induced from stem cells such as bone marrow precursors or iPSC.
  • the macrophage or macrophage precursor (a monocyte) is isolated from a supply tissue such as, but not limited to blood, lymph, bone marrow) and then subjected to in vitro cell or tissue culture to induce the desired tissue niche directed phenotype.
  • the cell composition is cryopreserved and/or contains a delivery agent.
  • macrophages may be generated in vitro from stem cells by various means. Macrophages generated from stem cells, such as BMSC, in the presence of IFNy or LPS are generally considered as “inflammatory” macrophages referred to as “M1 macrophages.” Those generated in the presence of IL-4 or IL-10 have what is called a “pro-resolution” activity and are referred to as “M2” macrophages.
  • the subject macrophage expresses M2 macrophage markers.
  • the macrophage cell expresses one or two or three or four or five of mmp9, arg2, mmp13a, L-plastin and cd163.
  • the macrophage cell expresses one or two or three or four or five of MRC1, CD200R, CD209, ARG1 and CHI3L3.
  • the macrophage subset expresses proxla and pou2f3.
  • the composition further comprises a stem cell and/or a macrophage cell.
  • the stem cell is a satellite cell. In another embodiment the stem cell is a unipotent or multipotent stem cell.
  • the active ingredient or main ingredient is, or is only, a fusion protein described herein.
  • the fusion protein described herein further comprises one or more moieties such as a linker, stability enhancing, signalling enhancing, delivery enhancing or label moiety.
  • compositions comprising a fusion protein, nucleic acid, vector, or cell as defined herein.
  • Pharmaceutical and physiologically active compositions are provided.
  • Cellular compositions are expressly provided.
  • the cell is a macrophage.
  • the macrophage is isolated from tissue.
  • the macrophage is induced from stem cells such as bone marrow precursors or iPSC.
  • the macrophage or macrophage precursor (a monocyte) is isolated from a supply tissue such as, but not limited to blood, lymph, bone marrow) and then subjected to in vitro cell or tissue culture to induce the desired tissue niche directed phenotype.
  • the cell composition is cryopreserved and/or contains a delivery agent.
  • macrophages may be generated in vitro from stem cells by various means. Macrophages generated from stem cells, such as BMSC, in the presence of IFNg or LPS are generally considered as “inflammatory” macrophages referred to as “M1 macrophages.” Those generated in the presence of IL-4 or IL-10 have what is called a “pro-resolution” activity and are referred to as “M2” macrophages.
  • the subject macrophage expresses M2 macrophage markers.
  • the macrophage cell expresses one or two or three or four or five of mmp9, arg2, mmp13a, L-plastin and cd163.
  • the macrophage cell expresses one or two or three or four or five of MRC1 , CD200R, CD209, Arg1 and Chi3l3.
  • the macrophage subset expresses proxla and pou2f3.
  • the composition comprises or is administered together with a supporting material such as a hydrogel, glue, foam or retentive material, scaffold etc.
  • a supporting material such as a hydrogel, glue, foam or retentive material, scaffold etc.
  • Delicate structures are generally suitable for enabling more delicate tissue regeneration.
  • materials can be used which are quite rapidly absorbed, such as certain fibrin, collagen, hydrogel and alginate formulations.
  • slowly absorbable synthetics can be used, such as poly-4-hydroxybutarate.
  • Silk fibres or even substantially smooth products derived from mammalian origin such as muscle extracellular matrix are also contemplated.
  • Non-absorbable synthetics, such as polypropylene and polyethylene provide support and reliability.
  • the composition comprises a fibrin hydrogel.
  • RAFT-acrylamide based support surfaces are provided to enhance tissue regeneration and bioavailability of a fusion protein, nucleic acid, vector, or cell to the target site.
  • composition comprising a fusion protein, nucleic acid, vector, or cell as described herein and any one or two or three or four of: (i) a satellite cell or precursor therefore or progeny thereof (ii) a macrophage or a precursor therefore or progeny thereof (iii) a scaffold (semi-solid or solid support) or retentive material (iv) a tissue delivery enhancing or cell retention moiety.
  • the scaffold or retentive material is a hydrogel, such as a fibrin or acrylamide hydrogel.
  • the tissue delivery enhancing or cell retention moiety is an ECM-binding moiety.
  • FIG. 1 Exemplary structure of an FC-NAMPT422-491 fusion protein as encompassed by the present invention. From N- to C-terminus, the structure comprises an IL-2 signalling sequence, a hinge region, an lgG1 Fc domain (with L234A, L235A, M252Y, S254T, T256E substitutions) and a NAMPTcif422-49i polypeptide linked to the C-terminus of the I gG 1 Fc domain via a (G4S)3 linker.
  • Figure 2 Human NAMPT variants stimulate human satellite cell proliferation.
  • FIG. 3 Human NAMPT variants stimulate human endothelial cell proliferation.
  • FIG. 1 Minimal versions of NAMPT protein enhance proliferation in response to muscle injury in zebrafish larvae. Treatment with lgG1-NAMPT422491 following needle-stick muscle injury induces a significant increase in cell proliferation within the injury zone as compared to 0.1% BSA negative control. Representative images are shown in panel (A).
  • the thick black lines and dashed black lines within the violin plot indicate the median and quartiles, respectively. Two-way ANOVA with Tuckey’s multiple comparison test.
  • FIG. 5 FC-NAMPT422-491 stimulates proliferation in zebrafish and retains wound site specificity.
  • A The effect on wound site (dotted line; skeletal muscle visualized by phalloidin staining) proliferation (Edll, white) was assayed following Fc and FC-NAMPT422-491 treatment in the Til zebrafish line. An individual example is provided for each group and these images are representative of the quantification presented in b. Scale bar, 50 pm.
  • FIG. 6 Fusion of lgG1 Fc to NAMPT422-491 does not interfere with the muscle restoration ability of NAMPT422-491.
  • Volumetric muscle loss defect (3 x 5 mm) was performed on hind limb rectus femoris muscle of 10-week-old C57BI/6 mice and treated with equimolar variants of NAMPT (0.5 pg; top right panel), NAMPT422-491 (0.096 pg; bottom left panel), IgGI Fc NAMPT422-491 (0.32 pg; bottom middle panel) or fibrin only (top middle panel) and lgG1-Fc (0.25 pg; bottom right panel) only as controls.
  • (A) Muscle regeneration evaluated at 10 days post injury by microCT expressed as percentage volume restored against contralateral healthy leg as reference (n 11 defects per group). Data are mean ⁇ SEM using. One-way ANOVA test with a Benjamin and Hochberg false rate discovery (FDR) was applied to compare all groups with a statistical significance threshold of 0.05.
  • Macrophage derived factors as detailed herein are proposed for use in modulating stem cell activity, in directly activating quiescent tissue stem cells and in tissue regeneration.
  • One macrophage derived factor described herein is Nicotinamide phosphoribosyltransferase (NAMPT, also known as visfatin and PBEF (pre-B cell enhancing factor). NAMPT has been identified by the inventors to be upregulated and produced by injury dwelling macrophages.
  • NAMPT Nicotinamide phosphoribosyltransferase
  • PBEF pre-B cell enhancing factor
  • the present invention provides a fusion protein comprising:
  • fusion proteins includes variants that activate quiescent tissue stem cells, and their derivatives comprising adaptations suitable for production, and clinical or commercial use, known in the art, such as enhanced tissue delivery or enhanced signalling functionalities.
  • the term includes orthologs and isoforms.
  • antibody refers to various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies.
  • antibody may also be used interchangeably with the term “immunoglobulin”.
  • a “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • recombinant human antibody includes all human sequence antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes; antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences.
  • recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.
  • Such antibodies can, however, be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • the term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the Fc region contains at least two heavy chain fragments comprising the CH2 and CH3 domains of an antibody.
  • the Fc region comprises two heavy chain fragments, preferably the CH2 and CH3 domains of said heavy chain.
  • the two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, s, Y, and p, respectively.
  • the fusion proteins of the invention comprise Fc regions which are not capable of homodimerisation.
  • Suitable technology for preventing homodimerisation of Fc regions of antibodies is well known and in some examples may include the use of “knob-into-hole” technology which enables the heterodimerisation of the Fc region of the fusion protein such that the “paired” Fc region does not comprise an NAM PT polypeptide or may comprise an alternative polypeptide fused thereto.
  • the Fc region comprises or consists of a hinge region combined with at least a portion of the heavy chain constant region, preferably the CH2 and CH3 domains of said heavy chain.
  • the hinge region may be derived from an immunoglobulin like molecule or non-immunoglobulin like molecule.
  • the hinge region comprises or consists of a hinge region from an lgG1, lgG2, lgG3 or lgG4, including a fragment thereof.
  • the fusion protein of the invention does not exhibit any effector function or any detectable effector function.
  • “Effector functions” or “effector activities” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibodydependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
  • CDC complement dependent cytotoxicity
  • ADCC antibodydependent cell-mediated cytotoxicity
  • phagocytosis phagocytosis
  • B cell receptor e.g. B cell receptor
  • Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).
  • Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95:652-656 (1998).
  • C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity.
  • a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101 :1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)).
  • FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12):1759-1769 (2006); WO 2013/120929 Al).
  • Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056).
  • Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).
  • an antibody variant may comprise an Fc region with one or more amino acid substitutions which diminish FcyR binding, e.g., substitutions at positions 234 and 235 of the Fc region (EU numbering of residues).
  • the substitutions are L234A and L235A (LALA) (See, e.g., WO 2012/130831).
  • an antibody variant may comprise an Fc region with one or more amino acid substitutions which increase the serum half-life of the Fc e.g., substitutions at positions 252, 254 and 256 of the Fc region (EU numbering of residues).
  • the substitutions are M252Y, S254T, T256E.
  • the antibody variant comprises an Fc region with L234A, L235A, M252Y, S254T and T256E substitutions.
  • alterations may be made in the Fc region that result in altered (i.e. , diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
  • CDC Complement Dependent Cytotoxicity
  • the Fc region includes mutations to the complement (C1q) and/or to Fc gamma receptor (FcyR) binding sites.
  • such mutations can render the fusion protein incapable of antibody directed cytotoxicity (ADCC) and complement directed cytotoxicity (CDC).
  • the Fc region as used in the context of the present invention does not trigger cytotoxicity such as antibody-dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC).
  • cytotoxicity such as antibody-dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC).
  • Fc region also includes native sequence Fc regions and variant Fc regions.
  • the Fc region may include the carboxyl-terminus of the heavy chain.
  • Antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain.
  • amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • Amino acid sequence variants of the Fc region of an antibody may be contemplated.
  • Amino acid sequence variants of an Fc region of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the Fc region of the antibody.
  • the Fc region of the antibody may be an Fc region of any of the classes of antibody, such as IgA, IgD, IgE, IgG, and IgM.
  • the “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
  • the antibody may be an Fc region of an IgG.
  • the Fc region of the antibody may be an Fc region of an lgG1, an lgG2, an lgG2b, an lgG3 or an lgG4.
  • the fusion protein of the present invention comprises an IgG of an Fc region of an antibody.
  • the Fc region of the antibody is an Fc region of an IgG, preferably lgG1.
  • the herein provided fusion proteins may comprise a linker (or “spacer”).
  • the polypeptide i.e. NAMPT cif polypeptide
  • a linker is usually a peptide having a length of up to 20 amino acids.
  • the term “linked to” or “fused to” refers to a covalent bond, e.g., a peptide bond, formed between two moieties. Accordingly, in the context of the present invention the linker may have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acids.
  • the herein provided fusion protein may comprise a linker between the NAMPT cif polypeptide and the Fc region of the antibody, such as between the C-terminus of the Fc regions and the N-terminus of the NAMPT cif polypeptide.
  • the herein provided fusion protein may comprise a linker between the NAMPT cif polypeptide and the Fc region of the antibody, such as between the N-terminus of the Fc regions and the C-terminus of the NAMPT polypeptide.
  • linkers have the advantage that they can make it more likely that the different polypeptides of the fusion protein fold independently and behave as expected.
  • the NAMPT polypeptide and the Fc region of an antibody may be comprised in a single-chain multi-functional polypeptide.
  • the fusion protein of the present invention includes a peptide linker.
  • the peptide linker links an NAMPT polypeptide with an Fc region of an antibody.
  • the peptide linker can include the amino acid sequence Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS, SEQ ID NO: 72) or Gly-Gly-Gly-Gly-Ser (GGGGS, SEQ ID NO: 73).
  • the peptide linker can include the amino acid sequence GGGGS (a linker of 6 amino acids in length) or even longer.
  • the linker may a series of repeating glycine and serine residues (GS) of different lengths, i.e., (GS)n where n is any number from 1 to 15 or more.
  • the linker may be (GS)3 (i.e., GSGSGS, SEQ ID NO: 74) or longer (GS)11 or longer.
  • n can be any number including 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more. Fusion proteins having linkers of such length are included within the scope of the present invention.
  • n is no more than 3 (ie such that when n equals 3 the linker is GSGSGS, SEQ ID NO: 74).
  • the linker comprises or consists of the sequence shown in SEQ ID NO: 41.
  • linkers include proline rich linkers such as the sequence PAPAP, APAPA and variations thereof.
  • the NAM PT polypeptide is directly fused to the Fc region of an antibody, such that there is no linker between the two regions of the fusion protein.
  • the fusion proteins described herein are proposed for use in stimulating wound healing and improving the quality of healing in order to promote full restoration of tissue function i.e., productive tissue repair and regeneration.
  • the present invention finds particular application in the treatment of, or muscle tissue regeneration in subject having, myopathies where inflammation is particularly undesirable.
  • the invention provides a method of stimulating muscle tissue regeneration in a subject where inflammation is undesirable, the method comprising administering to a muscle an effective amount of a fusion protein, composition or cell as described herein, wherein the fusion protein, composition or cell as described herein, thereby stimulating muscle tissue regeneration in this subject.
  • Reference to “regeneration” in relation to a muscle is used herein in a broad context and includes the flow on effects on muscle and muscle associated tissue as a direct result of muscle stem cell (also called satellite cell) activation.
  • regeneration includes muscle wound repair and muscle maintenance, growth, repair, augmentation of the ability of muscle cells to productively proliferate and form functional tissues.
  • the term includes generation of muscle tissue, and repair of an injured muscle, and pertains to the process of muscle regeneration (myogenesis) commencing with activation and proliferation of muscle stem cells, proliferation of myoblasts, early differentiation into myocytes and terminal differentiation into myofibres.
  • regeneration is associated with minimal fibrosis which allows for establishment of native structures or regenerated tissue having normal or approximating normal biological properties rather than fibrotic or weakened tissue.
  • Muscle functional properties may be determined by standard tests of contractile muscle function, including tests for strength (for example eccentric muscle contraction), power and endurance, as well as physical length and volume. The term also includes growth of muscle tissue in commercial cultures.
  • the inflammation that is undesirable is inflammation mediated by TLR activation, preferably TLR4 activation.
  • the subject may be diagnosed with an inflammatory myopathy.
  • inflammatory myopathies include polymyositis, dermatomyositis, inclusion body myositis, necrotizing autoimmune myopathy.
  • Other inflammatory myopathies, their clinical features and methods of diagnosing them are described in Dalakas, 2015, N Engl J Med 2015;372:1734-47.
  • the present invention provides a method of treating an inflammatory myopathy in a subject, the method comprising administering to a muscle of the subject an effective amount of a fusion protein, composition or cell as described herein, thereby treating an inflammatory myopathy.
  • the inflammatory myopathy is polymyositis, dermatomyositis, inclusion body myositis or necrotizing autoimmune myopathy.
  • the application enables a pharmaceutical or physiologically active regenerative composition comprising one or two or three or four or five of
  • the fusion protein in monomeric or dimeric form is modified to make the fusion protein suitable for attachment to a biological carrier or to the extracellular matrix.
  • the fusion protein is modified to enhance signalling through the CCR5 receptor by addition of moieties that bind co-receptors such as heparin sulphate proteoglycans (such as syndecans).
  • a fusion protein, nucleic acid, vector, cell or composition described herein are for use, or for use in manufacturing compositions for use, in stimulating muscle regeneration in vitro, ex vivo or in vitro.
  • compositions described herein are for use or when used in artificial muscle production (such as fish, bird or other non-human animal muscle for direct or indirect consumption).
  • artificial muscle production such as fish, bird or other non-human animal muscle for direct or indirect consumption.
  • supplementation to growth media enables scalability and more efficient muscle proliferation.
  • compositions described herein are for use or when used in stem cell therapy.
  • the compositions support expansion in vitro and/or are included in a transplant (or as a pre-treatment) to promote in vivo expansion and tissue integration.
  • the present application provides a method of stimulating tissue regeneration, the method comprising administering to an isolated or tissueresident tissue stem cell or a precursor thereof an effective amount of a composition comprising or encoding a fusion protein described herein, and optionally a component that enhances delivery to the tissue, wherein the fusion protein binds to tissue stem cells or their precursors and stimulates (activates) quiescent tissue stem cell proliferation and tissue regeneration.
  • the fusion protein comprises a component or moiety that enhances delivery to the target tissue.
  • compositions described herein are for use or when used, or for use in manufacturing compositions for use, in treating a muscular, neuromuscular, or musculoskeletal deficiency, disorder or injury.
  • Muscular, neuromuscular, or musculoskeletal deficiencies, disorders or injuries are known in the art. Deficiencies and disorders are found, for example, and without limitation in sarcopenia, cachexia and the muscular dystrophies, muscle atrophy, muscle pseudo hypertrophy or muscle dystrophy conditions and myopathies. All appropriate formats such as Swiss-style use, method of treatment and/or European-style use claims are encompassed.
  • isolated cell refers to a cell that has been removed from an organism in which it was originally found or a descendant of such a cell.
  • the cell may have been cultured in vitro, e.g., in the presence of other cells.
  • the cell may be destined to later be introduced into a second organism or re-introduced into the organism from which it (or the cell from which it is descended) was isolated.
  • isolated population of cells refers to a population of cells that has been removed and separated from a mixed or heterogeneous population of cells.
  • an isolated population is a substantially pure population of cells as compared to the heterogeneous population from which the cells were isolated or enriched.
  • the application enables a pharmaceutical or physiologically active regenerative composition comprising one or two or three or four or five of:
  • tissue stem cell such as a satellite cell
  • precursor therefore or progeny thereof
  • the present application provides cellular compositions comprising one or more of one or more stem cells, stromal cells, pre-satellite cells or satellite cells, pre-macrophages or macrophages or macrophage derived factors as described herein.
  • multipotent “tissue stem cell” include a premuscle cell or any pre-macrophage cells from which these cell may be produced in an essentially native form or modified to express heterologous or autologous factors.
  • the term multipotent “tissue stem cell” may include activated progeny of the tissue stem cell.
  • Tissue stem cells including muscle stem cells can be isolated (for ex vivo or in vitro or in vivo procedures) or induced.
  • a stem cell can be contacted with a media or composition comprising a fusion protein, nucleic acid, vector, or cell for any amount of time.
  • a stem cell can be contacted with a fusion protein, nucleic acid, vector, or cell for 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week or more.
  • the stem cell can be induced or stimulated to differentiate into a cell lineage selected from the group consisting of mesoderm, endoderm, ectoderm, neuronal, mesenchymal, and hematopoietic lineage.
  • the stem cell is a human stem cell, a multipotent adult stem cell, a pluripotent adult stem cell or an embryonic stem cell.
  • Human adult stem cells are mitotic and typically one daughter cell remains a stem cell.
  • Adult tissue comprises one or more resident committed progenitor or stem cells that occupy a specific niche in their tissue and actively sense and respond to their local environment. Each tissue typically has its own resident committed stem cell committed to producing progeny that differentiate into a specific range of cell types.
  • Muscle tissue comprises satellite cells to are committed to producing myoblasts.
  • Other well studied stem cells of this type are mesenchymal stem cells (MSC) that produce many different cell types inter alia muscle, cartilage, bone, fat, and haematopoietic stem cells (HSC) that produce all blood cells and the haematopoietic system, and neural stem cells (NSC).
  • MSC mesenchymal stem cells
  • HSC haematopoietic stem cells
  • NSC neural stem cells
  • MSC multipotent cell that refers to a cell that is able to differentiate into some but not all of the cells derived from all three germ layers. Thus, a multipotent cell is a partially differentiated cell.
  • MSC for example can be obtained by a number of methods well known in the art. See LISPN 5,486,358; 6,387,367; and LISPN 7,592,174, and LISPN 2003/0211602. MSC may be derived from bone, fat and other tissues where they reside. “Derived” from does not refer to direct derivation and merely indices where they were originally derived.
  • the stem cell is a non-embryonic or adult multipotent stem cell.
  • the stem cell is a HSC or MSC.
  • CCR5 can be stimulated to undergo differentiation by exposure to a fusion protein, nucleic acid, vector, or cell described herein. Cells are monitored to changes in expression of for example myogenic regulatory factors known in the art.
  • Cells may be cultured in standard media or specifically defined media.
  • Cell expression may be modified by techniques know in the art.
  • An induced or partially induced pluripotent stem cell is a convenient source of stem cells. These are derivable from a differentiated adult cell, such as human foreskin cells.
  • Human iPS cells can be generated by introducing specific sets of reprogramming factors into a non-pluripotent cell which can include, for example, Oct3/4, Sox family transcription factors (e.g., Sox1, Sox2, Sox3, Sox15), Myc family transcription factors (e.g., c-Myc, 1-Myc, n-Myc), Kruppel-like family (KLF) transcription factors (e.g., KLF1, KLF2, KLF4, KLF5), and/or related transcription factors, such as NANOG, LIN28, and/or Glisl.
  • the reprograming factors can be introduced into the cells using one or more plasmids, lentiviral vectors, or retroviral vectors.
  • the vectors integrate into the genome and can be removed after reprogramming is complete. In some cases, the vectors do not integrate (e.g., those based on a positive-strand, single-stranded RNA species derived from non-infectious (non-packaging) self-replicating Venezuelan equine encephalitis (VEE) virus, Simplicon RNA Reprogramming Kit, Millipore, SCR549 and SCR550).
  • VEE Venezuelan equine encephalitis
  • the Simplicon RNA replicon is a synthetic in vitro transcribed RNA expressing all four reprogramming factors (OKG-iG; Oct4, Klf4, Sox2, and Glisl) in a polycystronic transcript that is able to self-replicate for a limited number of cell divisions.
  • Human induced pluripotent stem cells produced using the Simplicon kit are referred to as “integration-free” and “footprint- free.”
  • Human iPS cells can also be generated, for example, by the use of miRNAs, small molecules that mimic the actions of transcription factors, or lineage specifiers.
  • Human iPS cells are characterized by their ability to differentiate into any cell of the three vertebrate germ layers, e.g., the endoderm, the ectoderm, or the mesoderm. Human iPS cells are also characterized by their ability propagate indefinitely under suitable in vitro culture conditions. Human iPS cells express alkaline phosphatase, SOX-2, OCT-4, Nanog and Tra-1-60 markers.
  • naive and primed iPS cells identify different pluripotency states of human iPS cells. Characteristics of naive and primed iPS cells are described in the art. Naive human iPS cells exhibit a pluripotency state similar to that of ES cells of the inner cell mass of a pre-implantation embryo. Such naive cells are not primed for lineage specification and commitment. Female naive iPS cells are characterized by two active X chromosomes. In culture, self-renewal of naive human iPS cells is dependent on leukemia inhibitory factor (LIF) and other inhibitors.
  • LIF leukemia inhibitory factor
  • Cultured naive human iPS cells display a clonal morphology characterized by rounded dome-shaped colonies and a lack of apico-basal polarity. Cultured naive cells can further display one or more pluripotency makers as described elsewhere herein. Under appropriate conditions, the doubling time of naive human iPS cells in culture can be between 16 and 24 hours.
  • Primed human iPSC express a pluripotency state similar to that of postimplantation epiblast cells. Such cells are primed for lineage specification and commitment.
  • Female primed iPSCs are characterized by one active X chromosome and one inactive X chromosome.
  • self-renewal of primed human iPSCs is dependent on factors such as fibroblast growth factor (FGF) and activin.
  • Cultured primed human iPSCs display a clonal morphology characterized by an epithelial monolayer and display apico-basal polarity. Under appropriate conditions, the doubling time of primed human iPSCs in culture can be 24 hours or more depending upon the level from the adult cells from which they were derived.
  • Embryonic stem cells are characteristically pluripotent i.e. , they have the capacity, under different conditions, to differentiate to cell types characteristic of all three germ cell layers (endoderm, mesoderm and ectoderm). Pluripotent cells are characterized primarily by their ability to differentiate to all three germ layers. In some embodiments, a pluripotent cell is an undifferentiated cell. Pluripotent cells also have the potential to divide in vitro for more than one year or more than 30 passages.
  • ESC are typically the pluripotent stem cells of the inner cell mass of the embryonic blastocyst (see U.S. Pat. Nos. 5,843,780, 6,200,806). Such cells can similarly be obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer (see U.S. Pat. Nos. 5,945,577, 5,994,619, 6,235,970).
  • Exemplary distinguishing embryonic stem cell characteristics include, without limitation, gene expression profile, proliferative capacity, differentiation capacity, karyotype, responsiveness to particular culture conditions.
  • the stem cell is adult.
  • the stem cells are autologous or heterologous to the subject.
  • the stem cell is mammalian or human.
  • Macrophages and stem cells including satellite cells may be prepared using art recognised methods and as described herein and include the use of iPSC and optionally gene editing procedures.
  • isolated macrophages or stem-cell derived macrophages are modified to express a polypeptide or fusion protein described herein.
  • M2 type macrophages are selected or provided.
  • stem cells are contacted with a fusion protein, nucleic acid, vector, or cell in vitro, ex vivo or in vivo as described herein to induce activation and proliferation.
  • Stem cells treated in vitro or ex vivo may be introduced into a wound site to effect repair or administered systemically to effect regeneration of damaged tissue or to treat or improve muscle related conditions as described herein.
  • a fusion protein, nucleic acid, vector, or cell expressing said fusion protein may be administered in the form of functionalized hydrogels either alone or together with cells for transplantation.
  • Such hydrogels or similar biomaterials or scaffolds provide enhanced transplantation efficiency at the wound site.
  • Hydrogels may be ECM based such as fibrin based.
  • hydrogels may be non-ECM based such as acrylamide based using RAFT technology (see Chiefari et al Macromol. 37:5559-5526, 1998 and Fairbanks et al Advanced Drug Delivery Reviews 91 141-152, 2015). Suitable materials regulate release kinetics, and have desired mechanical and physical properties for tissue regeneration as known in the art.
  • satellite cells are encapsulated in CCR5 functionalised hydrogels or other biomaterials.
  • Kits comprising the cellular compositions and/or agents described herein are also provided. Kits suitable for muscle repair or regeneration are specifically contemplated.
  • the fusion protein, nucleic acid, vector, or cell can be pre-formulated for administration or ingredients for formulation can be provided with the kit.
  • the fusion protein, nucleic acid, vector, or cell is for example formulated in a hydrogel or other supporting vehicle for topical application.
  • the fusion protein, nucleic acid, vector, or cell may be, for example, lyophilised or liquid.
  • protein polypeptide
  • polypeptide polymeric forms of amino acids of any length unless that length is defined, including coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids.
  • the terms also include polymers that have been modified, such as polypeptides having modified peptide backbones.
  • Proteins are said to have an “N-terminus” be “N-terminal” and to have a “C- terminus” or be “C-terminal.”
  • N-terminus relates to the start of a protein or polypeptide, terminated by an amino acid with a free amine group (--NH2).
  • C- terminus relates to the end of an amino acid chain (protein or polypeptide), which is in nature terminated by a free carboxyl group (--COOH).
  • reference to C-terminal and N-terminal fragments broadly describe the region of the full length molecule from which the elected part is derived and it excludes a full length or a native molecule.
  • a C-terminal fragments does not have to but may include all the C- terminal amino acids and N-terminal fragments do not have to but may include all the N- terminal amino acids.
  • the application discloses and enables the use of a range of fusion proteins, nucleic acids, vectors, or cells based upon the initial findings described in the examples.
  • a number of peptide modifications are known in the art to stabilise peptides against serum proteases or to promote intracellular positioning and these are encompassed. Some such modified peptides can be expressed from nucleic acids in a cell, others are manufactured synthetically. Similarly, where it is desirable to target the fusion protein described herein to one or more specific cell types, this may be achieved either by ex vivo manipulation of target cells, or incorporation of targeting moieties able to bind specifically to target cells or tissues such as ECM binding moieties, as known in the art.
  • a “conservative amino acid substitution” is one in which the naturally or non- naturally occurring amino acid residue is replaced with a naturally or non-naturally occurring amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., Lys, Arg, His
  • acidic side chains e.g., Asp, Glu
  • uncharged polar side chains e.g., Gly, Asn, Gin, Ser, Thr, Tyr, Cys
  • nonpolar side chains e.g., Ala, Vai, Leu, lie, Pro, Phe, Met, Trp
  • beta-branched side chains e.g., Thr, Vai, lie
  • aromatic side chains e.g., Phe, Trp, His
  • a predicted nonessential amino acid residue in a fusion protein as described herein, for example may be replaced with another amino acid residue from the same side chain family.
  • substitutions based on isosteric considerations e.g. norleucine for methionine
  • other properties e.g. 2-thienylalanine for phenylalanine.
  • a full amino acid sub-classification is set out in Table 2 and exemplary substitutions are set out in Table 3.
  • Trp residues are substituted.
  • a fusion protein described herein may comprise modifications known to modify the pharmacokinetic features of peptides, such as by increasing protease resistance in vivo.
  • the peptide comprises one or more of a linker or spacer such as GGS or repeats of GGS and variants known in the art), a modified or nonnatural or non-proteogenic amino acid, a modified side-chain, a modified backbone, terminal modified groups or comprises a modified spatial constraint or is a D-retro- inverso peptide.
  • the peptide is a pseudopeptide, peptoid, azapeptide, cyclized, stapled, ether or lactam peptide or comprises a spatial constraint.
  • the fusion protein described herein is conjugated or otherwise attached/bound/expressed with as appropriate to a lipid, carbohydrate, polymer, protein, nanoparticle, peptide, proteoglycan, antibody or fragment or antigen binding form thereof, aptamer, or nucleic acid.
  • the fusion protein described herein specifically binds to muscle cells or muscle cell tissue or associated structures eg, ECM.
  • the fusion protein described herein includes physiologically or pharmaceutically acceptable salts, hydrates, stereoisomers, and prodrugs.
  • non-essential amino acids may be altered.
  • Reference to “non-essential” amino acid residue means a residue that can be altered from the wildtype sequence of a polypeptide without abolishing or substantially altering its ability to bind to an endogenous or heterologous CCR5.
  • the fusion protein described herein comprises or encodes an amino acid sequence having 1 , 2, 3, 4, 5 or 6 conservative (for example those outlined in Table 3 above) or non-conservative amino acid substitution, deletion or addition to the above sequences but retains CCR5 interacting activity.
  • the invention also provides a nucleic acid molecule from which the fusion protein described herein is expressible. Nucleotide sequences encoding fusion proteins described herein are herein disclosed.
  • the nucleic acid molecule is an RNA or DNA or RNA : DNA or a chemically modified form thereof.
  • a proportion of at least one type of nucleotide is chemically modified to increase its stability in vivo.
  • nucleotide e.g, cysteine and/or uracil
  • the nucleic acid is in the form of a viral or non-viral vector.
  • the fusion protein, nucleic acid, vector, cell or composition described herein is administered to cells ex vivo.
  • the present invention encompasses the use of genetically modified cell depots (e.g. genetically modified macrophage, etc).
  • the fusion protein further comprises an antibody or antibody fragment that targets the agent specifically to target cells, such as muscle stem cells.
  • the present application provides a pharmaceutical or physiological composition comprising a fusion protein, nucleic acid, vector or cell as defined herein above.
  • the application enables a method of treating a muscle injury or a person with a diminished or suboptimal ability to repair or regenerate muscle, comprising administering to the subject an effective amount of a composition comprising a fusion protein, nucleic acid, vector or cell, or composition comprising a fusion protein, nucleic acid, vector or cell, sufficient to stimulate muscle stem cell proliferation and muscle regeneration.
  • compositions include physiologically or pharmacologically or pharmaceutically acceptable vehicles that are not biologically or otherwise undesirable.
  • Pharmacologically acceptable salts, esters, pro-drugs, or derivatives of a compound described here is a salt, ester, pro-drug, or derivative that is not biologically or otherwise undesirable.
  • the fusion protein is modified. Fusion protein activity are tolerant to additional moieties, flanking residues and substitutions within the defined boundaries. Similarly backbone modifications and replacements, side-chain modifications and N and C-terminal modifications are conventional in the art. Generally, the modification is to enhance stability or pharmacological profile, targeting/delivery. For example, peptide cyclisation or stapling is conventional for enhancing peptide stability. In another embodiment, peptides or agents are in the form of micro or nano-particles or bubbles, gels, liposomes, conjugates or fusion proteins comprising moieties adapted for stability, delivery or specificity to the target tissue.
  • fusion proteins or their encoding nucleic acids where appropriate are assembled in liposomes, hydrogels, emulsions, viral vectors, viral-like particles or virosomes.
  • specific binding moieties such as antibody or antibody fragments or mimics are used to target fusion proteins to the muscle environment.
  • compositions generally comprise a fusion protein, peptidomimetic or an encoding nucleic acid where appropriate, and a pharmaceutically acceptable carrier and/or diluent.
  • the carrier may be a nanocarrier.
  • the fusion proteins of the present disclosure are not naturally occurring molecules, but instead are modified forms of naturally occurring molecules which do not possess certain features or functions of the naturally occurring full length molecules.
  • NAM PT enzymatic activity may be absent.
  • the fusion protein described herein is constrained by means of a linker which is covalently bound to at least two amino acids in the peptide.
  • a linker which is covalently bound to at least two amino acids in the peptide.
  • the fusion protein described herein is delivered in the form of nucleic acid molecules encoding same or pro-drugs thereof or vectors comprising nucleic acid molecules encoding same or pro-drugs thereof.
  • the nucleic acid is mRNA.
  • the fusion protein described herein may bind the surface of the muscle stem cell or function internally to stimulate signalling and proliferation.
  • the fusion protein, nucleic acid, vector or cell described herein is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregates as micelles, insoluble monolayers, liquid crystals or lamellar layers in aqueous solution.
  • amphipathic agents such as lipids which exist in aggregates as micelles, insoluble monolayers, liquid crystals or lamellar layers in aqueous solution.
  • the disclosure enables a composition comprising a fusion protein as described herein which interacts with endogenous CCR5 proteins for use as a medicament or for use in therapy.
  • the present disclosure enables a composition for stimulating muscle stem cell proliferation comprising a fusion protein as described herein or a nucleic acid molecule from which the peptide is expressible.
  • the subject composition is co-administered with a second physiologically active, therapeutic or prophylactic or regenerative agent.
  • cytokines include without limitation one or more of IGF-1, TGF-p, GDF-5, bFGF, PDGF- b3, IL-4.
  • the present disclosure provides for the use of the fusion protein, nucleic acid, vector or cell as described herein in the manufacture of a medicament for stimulating muscle regeneration or in stem cell therapy.
  • amino acid includes naturally occurring amino acids or non- naturally occurring amino acids.
  • Peptide compounds are generally and conventionally modifiable by addition of moieties, flanking peptide residues, and substitutions within understood parameters. Peptides can furthermore comprise routine modified backbones, side chains, peptide bond replacements, and terminal modifications using standard peptide chemistries.
  • amino acids incorporated into the amino acid sequence described herein may be L-amino acids, D-amino acids, L- p -homo amino acids, D- p -homo amino acids or N-methylated amino acids, sugar amino acids, and/or mixtures thereof.
  • Non-natural amino acids may not be recognised by proteases and may therefore alter the half-life.
  • the D-retro inversion sequence is employed.
  • Non-naturally occurring amino acids include chemical analogues of a corresponding naturally occurring amino acid.
  • unnatural amino acids and derivatives include, but are not limited to, 4-amino butyric acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine, nor leucine, norvaline, phenylglycine, ornithine, sarcosine, 2-thienyl alanine and/or D-isomers of amino acids.
  • amino acid analog refers to a molecule which is structurally similar to a naturally occurring amino acid and which can be substituted for an amino acid in the formation of a peptidomimetic macrocycle.
  • Amino acid analogs include, without limitation, compounds which are structurally identical to an amino acid, as defined herein, except for the inclusion of one or more additional methylene groups between the amino and carboxyl group or for the substitution of the amino or carboxy group by a similarly reactive group (e.g., substitution of the primary amine with a secondary or tertiary amine, or substitution or the carboxy group with an ester).
  • Linkers or spacers may be amino acids or nucleic acids or other atomic structures known in the art, typically between 2 and 10 amino acids or nucleotides in length. Spacers should be flexible enough to allow correct orientation of a fusion protein as described herein, such as those including nanoparticles, antibody fragments, liposomes, cell penetrating and/or intracellular delivery moieties.
  • One form of spacer is the hinge region from IgG suitable for use when the construct comprises an antigen binding moiety for cellular targeting.
  • a fusion protein described herein is conjugated or otherwise associated (covalent or non-covalent attachment) with a delivery agent.
  • the delivery agent delivers the peptide to tissue, a target cell or cell population.
  • Derivatives of fusion proteins described herein include biologically active fragments thereof as described herein comprising the structures described or orthologs.
  • Derivatives also include molecules having a percent amino acid or polynucleotide sequence identity over a window of comparison after optimal alignment. In one embodiment the percentage identity is at least 80%-99% including any number in between 80 and 99.
  • Suitable assays for the biological activity of fusion proteins are known to the skilled addressee and are described in the specification.
  • markers of fusion protein activity include upregulation of satellite cell signalling (eg, MAPK), stem cell and myoblast proliferation and differentiation.
  • the fusion protein is modified with a moiety which is not a naturally occurring amino acid residue.
  • the moiety may be selected from the group consisting of a detectable label, a non-naturally occurring amino acid as described herein, a reactive group, a fatty acid, cholesterol, a lipid, a bioactive carbohydrate, a nanoparticle, a small molecule drug, and a polynucleotide.
  • the moiety is a detectable tag label.
  • the detectable label is selected from the group consisting of a fluorophore, a fluorogenic substrate, a luminogenic substrate, and a biotin.
  • affinity agents and moieties for detection include fluorescent and luminescent compounds, metals, dyes.
  • Other useful moieties include affinity tags, biotin, lectins, chelators, lanthanides, fluorescent dyes, FRET acceptor/donors.
  • the fusion protein which may comprise a detectable label, is accompanied in a kit with a modified control version of the fusion protein wherein the conserved residues of the fusion protein are substituted with for example alanine.
  • Kits comprising the agents are proposed for sale and may be used for screening purposes or therapeutic purposes.
  • Peptides of this type may be obtained through the application of recombinant nucleic acid techniques as, for example, described in Sambrook et al. MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbour Press, 1989), in particular Sections 16 and 17; Ausubel eta/CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons, Inc. 1994-1998), in particular Chapters 10 and 16; and Coligan etal. CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5 and 6.
  • peptides of this type may be synthesised using conventional liquid or increasingly solid phase synthesis techniques.
  • initial reference may be made to solution synthesis or solid phase synthesis as described, for example, by Atherton and Sheppard in SOLID PHASE PEPTIDE SYNTHESIS: A PRACTICAL APPROACH (IRL Press at Oxford University, Oxford, England, 1989), see particularly Chapter 9, or by Roberge etal. (1995 Science 269: 202).
  • Azapeptide synthesis was previously hampered by tedious solution-phase synthetic routes for selective hydrazine functionalization. Recently, the submonomer procedure for azapeptide synthesis, has enabled addition of diverse side chains onto a common semicarbazone intermediate, providing a means to construct azapeptide libraries by solution- and solid-phase chemistry. In brief, aza residues are introduced into the peptide chain using the submonomer strategy by semicarbazone incorporation, deprotonation, N-alkylation, and orthogonal deprotection. Amino acylation of the resulting semicarbazide and elongation gives the desired azapeptide.
  • Azapeptide analogues of growth hormone-releasing peptide-6 have for example been pursued as ligands of the cluster of differentiation 36 receptor (CD36) and show promising activity for the development of treatments for angiogenesis-related diseases, such as age-related macular degeneration, as well as for atherosclerosis.
  • Azapeptides have also been employed to make a series of conformationally constrained second mitochondria-derived activator of caspase (Smac) mimetics that exhibit promising apoptosis-inducing activity in cancer cells.
  • cyclic azapeptide derivatives were used to make an aza scan to study the conformation-activity relationships of the anticancer agent cilengitide, cyclo(RGDf- N(Me)V), and its parent counterpart cyclo(RGDfV), which exhibit potency against human tumor metastasis and tumor-induced angiogenesis.
  • Innovations in the synthesis and application of azapeptides are described in Acc Chem Res. 2017 Jul 18;50(7):1541-1556.
  • peptides can be produced by digestion of an adaptor polypeptide with proteinases such as endoLys-C, endoArg-C, endoGlu-C and staphylococcus V8- protease.
  • the digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques. Measures that may be taken to optimize pharmacodynamics parameters of peptides and peptide analogs are described by Werle M. etal (2006) Strategies to improve plasma half-life time of peptide and protein drugs amino Acids 30(4): 351-367; and Di L (2014) Strategic approaches to optimising peptide ADME properties AAPS J 1-10.
  • the fusion protein may be stabilised for example via nanoparticles, liposomes, micelles or for example PEG as known in the art. Methods to form liposomes are described in: Prescott, Ed. Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq., the contents of which is incorporated herein by reference. Polymer nanoparticles ideally use surfactants that are not toxic or physically adsorbed to the nanoparticle. In one aspect, biodegradable surfmers are used.
  • biodegradable, poly(ethylene glycol) (PEG)ylated N-(2-hydroxypropyl) methacrylamide (HPMA) based surfmers are synthesized and used to stabilize lipophilic NPs.
  • the NP core is made from a macromonomer comprising a poly(lactic acid) (PLA) chain functionalized with HPMA double bond.
  • the nanoparticle forming polymer chains are then constituted by a uniform poly(HPMA) backbone that is biocompatible and water soluble and hydrolysable PEG and PLA pendants assuring the complete degradability of the polymer.
  • the stability provided by the synthesized surfmers is studied in the cases of both emulsion free radical polymerization and solution free radical polymerization followed by the flash nanoprecipitation of the obtained amphiphilic copolymers.
  • stabilising or heterologous moieties include NMEG, ECM binding, syndecan binding albumin, albumin binding proteins, immunoglobulin Fc domain.
  • nanoparticles comprising the fusion protein, nucleic acid, vector or cell can be further modified by the conjugation of tissue type specific binding agents, antibodies or fragments thereof known in the art.
  • Fusion proteins described herein may be administered in a range of retention enhancing compositions known in the art, such as gels, foams, glues, hydrogels, patches, and films, and the like.
  • Oral and injectable solution solubilizing excipients include water-soluble organic solvents (polyethylene glycol 300, polyethylene glycol 400, ethanol, propylene glycol, glycerin, N-methyl-2-pyrrolidone, dimethylacetamide, and dimethylsulfoxide), non-ionic surfactants (Cremophor EL, Cremophor RH 40, Cremophor RH 60, d-a- tocopherol polyethylene glycol 1000 succinate, polysorbate 20, polysorbate 80, Solutol HS 15, sorbitan monooleate, poloxamer 407, Labrafil M-1944CS, Labrafil M-2125CS, Labrasol, Gellucire 44/14, Softigen 767, and mono- and di-fatty acid esters of PEG 300, 400, or 1750), water-insoluble lipids (castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil
  • a construct or vector for expressing a fusion protein described herein from a recipient cell can comprise one or more DNA regions comprising a promoter operably linked to a nucleotide sequence encoding the peptide.
  • the promoter can be inducible or constitutive.
  • Suitable constitutive promoters include, e.g., an immediate early cytomegalovirus (CMV) promoter, an Elongation Growth Factor - la (EF-la) gene promoter, a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycl
  • the expression constructs may be generated by any suitable method including recombinant or synthetic techniques, utilizing a range of vectors known and available in the art such as plasmids, bacteriophage, baculovirus, mammalian virus, artificial chromosomes, among others.
  • the expression constructs can be circular or linear, and should be suitable for replication and integration into eukaryotes.
  • Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses and lentiviruses.
  • retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to the subject stem cells.
  • retroviral systems are known in the art.
  • the nucleic acid may be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular (e.g., by use of a retroviral vector, by direct injection, by use of microparticle bombardment, by coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide or other intracellular targeting moiety.
  • a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression.
  • nucleic acid and “polynucleotide,” used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. They include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.
  • Nucleic acids are said to have “5’ ends” and “3’ ends” because mononucleotides are reacted to make oligonucleotides in a manner such that the 5’ phosphate of one mononucleotide pentose ring is attached to the 3’ oxygen of its neighbor in one direction via a phosphodiester linkage.
  • An end of an oligonucleotide is referred to as the “5’ end” if its 5’ phosphate is not linked to the 3’ oxygen of a mononucleotide pentose ring.
  • An end of an oligonucleotide is referred to as the “3’ end” if its 3’ oxygen is not linked to a 5’ phosphate of another mononucleotide pentose ring.
  • a nucleic acid sequence even if internal to a larger oligonucleotide, also may be said to have 5’ and 3’ ends.
  • discrete elements are referred to as being “upstream” or 5’ of the “downstream” or 3’ elements.
  • Codon optimization may be used and generally includes a process of modifying a nucleic acid sequence for enhanced expression in particular host cells by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the genes of the host cell while maintaining the native amino acid sequence.
  • a nucleic acid encoding a Cas protein can be modified to substitute codons having a higher frequency of usage in a given prokaryotic or eukaryotic cell, including a bacterial cell, a yeast cell, a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, a hamster cell, or any other host cell, as compared to the naturally occurring nucleic acid sequence.
  • Codon usage tables are readily available, for example, at the “Codon Usage Database.” These tables can be adapted in a number of ways. See Nakamura etal. (2000) Nucleic Acids Research 28:292, herein incorporated by reference in its entirety for all purposes. Computer algorithms for codon optimization of a particular sequence for expression in a particular host are also available (see, e.g., Gene Forge).
  • a nucleic acid molecule as described herein may in any form such as DNA or RNA, including in vitro transcribed RNA or synthetic RNA.
  • Nucleic acids include genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules and modified forms thereof.
  • a nucleic acid molecule may be single stranded or double stranded and linear or closed covalently to form a circle.
  • the RNA may be modified by stabilizing sequences, capping, and polyadenylation.
  • RNA or DNA and may be delivered as plasmids to express the peptide.
  • RNA-based approaches are routinely available.
  • RNA relates to a molecule which comprises ribonucleotide residues and preferably being entirely or substantially composed of ribonucleotide residues.
  • “Ribonucleotide” relates to a nucleotide with a hydroxyl group at the 2’-position of a -D- ribofuranosyl group.
  • the term includes double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • Such alterations can include addition of non-nucleotide material, such as to the end(s) of a RNA or internally, for example at one or more nucleotides of the RNA.
  • Nucleotides in RNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.
  • An optimised mRNA based composition could comprise a 5’ and 3’ non translated region (5’-UTR, 3’-UTR) that optimises translation efficiency and intracellular stability as known in the art.
  • removal of uncapped 5 ‘-triphosphates can be achieved by treating RNA with a phosphatase.
  • RNA may have modified ribonucleotides in order to increase its stability and/or decrease cytotoxicity.
  • 5-methylcytidine is substituted partially or completely, for cytidine.
  • the term “modification” relates to providing an RNA with a 5’-cap or 5’-cap analog.
  • 5’-cap refers to a cap structure found on the 5’-end of an mRNA molecule and generally consists of a guanosine nucleotide connected to the mRNA via an unusual 5’ to 5’ triphosphate linkage. In one embodiment, this guanosine is methylated at the 7-position.
  • conventional 5’- cap refers to a naturally occurring RNA 5’-cap, preferably to the 7-methylguanosine cap.
  • the term “5’-cap” includes a 5’-cap analog that resembles the RNA cap structure and is modified to possess the ability to stabilize RNA and/or enhance translation of RNA.
  • RNA with a 5’-cap or 5’-cap analog may be achieved by in vitro transcription of a DNA template in the presence of said 5’-cap or 5’-cap analog, wherein said 5’-cap is co- transcriptionally incorporated into the generated RNA strand, or the RNA may be generated, for example, by in vitro transcription, and the 5’-cap may be attached to the RNA post-transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.
  • capping enzymes for example, capping enzymes of vaccinia virus.
  • RNA may be an extension or truncation of the naturally occurring poly(A) tail or an alteration of the 5’- or 3 ‘-untranslated regions (UTR) such as introduction of a UTR which is not related to the coding region of said RNA, for example, the exchange of the existing 3’-UTR with or the insertion of one or more, preferably two copies of a 3’-UTR derived from a globin gene, such as alpha2- globin, alphal-globin, beta-globin.
  • RNA having an unmasked poly-A sequence is translated more efficiently than RNA having a masked poly-A sequence.
  • RNA In order to increase stability and/or expression of the RNA it may be modified so as to be present in conjunction with a poly-A sequence, preferably having a length of 10 to 500, more preferably 30 to 300, even more preferably 65 to 200 and especially 100 to 150 adenosine residues.
  • a poly-A sequence preferably having a length of 10 to 500, more preferably 30 to 300, even more preferably 65 to 200 and especially 100 to 150 adenosine residues.
  • Modified mRNA may be synthesised enzymatically and packaged into nanoparticles such as lipid nanoparticles and administered, for example intramuscularly.
  • the nucleic acid molecule can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions.
  • colloidal drug delivery systems e.g., liposomes, microspheres, microemulsions, nanoparticles and nanocapsules
  • macroemulsions e.g., liposomes, microspheres, microemulsions, nanoparticles and nanocapsules
  • Targeted delivery of agents to particular cell subsets can enhance the therapeutic index.
  • Ex vivo approaches contemplate the administration of gene editing such as CRISPR components to modify cells to contain or express a fusion protein as described herein.
  • the fusion proteins, nucleic acids, vectors, or cells as described herein, or compositions comprising or encoding the same can be administered to patients for wound healing or to delay, maintain, or regenerate muscle in various conditions associated with muscle loss or diminished ability to regenerate functionally.
  • the fusion proteins, nucleic acids, vectors, or cells as described herein, or compositions comprising or encoding the same may be delivered by injection, by topical or mucosal application, by inhalation or via oral route including modified release modes, over periods of time and in amounts which are effective to stimulate muscle regeneration levels in a subject.
  • Administration may be topical or systemic (e.g., parenteral v/a for example intravenous, intraperitoneal, intradermal, sub cutaneous or intramuscular routes) or targeted.
  • administration of CCR5- interacting agent is systemic or directly to a wound.
  • Sub cutaneous or intramuscular routes may be directly to an affected muscle tissue.
  • a fusion protein, nucleic acid, vector, or cell as described herein, or compositions comprising or encoding the same can be formulated in the form of ointments, creams, patches, powders, or other formulations suitable for topical formulations. Small molecular weight polypeptide or fusion protein formulations can deliver the agent from skin to deeper muscle tissue. Accordingly, such formulations may comprise one or more agents that enhance penetration of active ingredient through skin.
  • a fusion protein, nucleic acid, vector, or cell as described herein, or compositions comprising or encoding the same can be included in wound dressings and/or skin coating compositions.
  • the amount of the agent to be administered may be determined by standard clinical techniques by those of average skill within the art.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed will also depend on the nature of the agent and other clinical factors (such as the condition of the subject their weight, age, other conditions, the route of administration and type of composition (cellular, scaffolded, hydrogel baes or oral formulations).
  • the precise dosage to be therapeutically or prophylactically effective and non-detrimental can be determined by those skilled in the art.
  • Pharmaceutical compositions are conveniently prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington, the Science and Practice of Pharmacy, 20 th Edition, Remington, J., ed. (2000) and later editions.
  • an effective amount includes a therapeutically or physiologically or regeneratively effective amount.
  • a “therapeutically-effective amount” as used herein means that amount of the composition comprising chemokine receptor agonist activity which is effective for producing some desired therapeutic effect in at least a subpopulation of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.
  • an amount of a polypeptide or fusion protein administered to a subject that is sufficient to produce a statistically significant, measurable muscle repair or regeneration. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
  • a therapeutically effective amount can vary with the subject’s history, age, condition, sex, as well as the severity and type of the medical condition in the subject, and administration of other pharmaceutically active agents.
  • administer refers to the placement of a composition into a subject by a method or route which results in at least partial localization of the composition at a desired site such that desired effect is produced.
  • Routes of administration suitable for the instant compositions with vary depending upon its format and include both local and systemic administration.
  • local administration results in more of the fusion proteins, nucleic acids, vectors, or cells as described herein, or compositions comprising or encoding the same, being delivered to a specific location as compared to the entire body of the subject, whereas, systemic administration results in delivery to essentially the entire body of the subject.
  • One method of local administration is by intramuscular injection.
  • the term “administering” also include transplantation of a cell into a subject.
  • transplantation refers to the process of implanting or transferring at least one cell into a subject.
  • the term “transplantation” includes, e.g., autotransplantation (removal and transfer of cell(s) from one location on a patient to the same or another location on the same patient), allotransplantation (transplantation between members of the same species), and xenotransplantation (transplantations between members of different species).
  • Skilled artisan is well aware of methods for implanting or transplantation of stem cells for muscle repair and regeneration, which are amenable to the present invention. See for example, U.S. Pat. No. 7,592,174 and U.S. Pat. Pub. No. 2005/0249731, content of both of which is herein incorporated by reference.
  • regeneration of muscle tissue by the present methods may be associated with minimal fibrosis.
  • the methods and agents described herein may reduce and/or inhibit formation of scar-like tissue in the damaged or non-regenerating or atrophying muscle tissue. Accordingly, in some embodiments, formation of scar-like tissue formation in the damaged muscle tissue is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% relative to a control without the present agents. Adipose deposition may be similarly reduced.
  • suitable dosage ranges for intravenous administration of a fusion protein, nucleic acid, vector, or cell as described herein, or compositions comprising or encoding the same are generally about 1.25 - 5 micrograms of active compound per kilogram (Kg) body weight.
  • Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral compositions preferably contain 10% to 95% active ingredient.
  • derivative is meant an agent or active that has been derived from the fusion protein by modification of the amino acid sequence, or, for example by conjugation or complexing or expression (eg, as a fusion protein) with other chemical moieties or by post-translational modification techniques as would be understood in the art.
  • derivative also includes within its scope alterations that have been made to a parent sequence including additions, or deletions that provide for functionally equivalent or functionally enhanced molecules.
  • isolated is meant material that is substantially or essentially free from components that normally accompany it in its native state.
  • Subject includes patient, and refers to any subject of medical or veterinary interest.
  • Subjects may be a vertebrate subject, such as mammalian subject (e.g, bovines, pigs, dogs, cats, equine, lama, camelids, etc.), non-mammals, reptiles, birds, fish.
  • the subject includes a human, for whom prophylaxis or therapy is desired.
  • the subject may be in need of prophylaxis or treatment for a cancer, wound care, sarcopenia or other pathology, disease, disorder or condition associated with tissue degeneration.
  • polynucleotide or “nucleic acid” as used herein designates mRNA, RNA, cRNA, cDNA or DNA.
  • the term typically refers to oligonucleotides greater than 30 nucleotides in length.
  • sequence “identity” as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base ⁇ e.g., A, T, C, G, II) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e.
  • sequence identity may be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for Windows; available from Hitachi Software Engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software.
  • Amino acid sequence identity may also be determined using the EMBOSS Pairwise Alignment Algorithms tool available from The European Bioinformatics Institute (EMBL-EBI), which is part of the European Molecular Biology Laboratory. This tool is accessible at the website located at www.ebi.ac.uk/Tools/emboss/align/.
  • This tool utilizes the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970). Default settings are utilized which include Gap Open: 10.0 and Gap Extend 0.5. The default matrix “Blosum62” is utilized for amino acid sequences and the default matrix.
  • sequence “similarity” refers to the percentage number of amino acids that are identical or constitute conservative amino acid substitutions as defined in Table 3 above. Similarity may be determined using sequence comparison programs such as GAP (Deveraux et al, 1984 Nucleic Acids Research 12: 387-395). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP. Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manual, 3 rd ed., vol. 1-3, ed.
  • the injury site was filled with fibrin hydrogel with or without NAM PT (0.5 pg), NAMPT422-491 (0.096 pg), IgGI Fc NAMPT422-491 (0.32 pg) (hydrogel components; 40 pl, 8 mg/ml human fibrinogen (FIB3, Enzyme Research Laboratories), 4 ll/rnl bovine thrombin (T4648, Sigma), 5 mM CaCI2, 17 pg/ml of aprotinin (ab146286, Abeam)) which polymerized in the defect. Then, the soft tissue was closed with stitches.
  • NAM PT 0.5 pg
  • NAMPT422-491 0.096 pg
  • IgGI Fc NAMPT422-491 (0.32 pg)
  • hydrogel components 40 pl, 8 mg/ml human fibrinogen (FIB3, Enzyme Research Laboratories), 4 ll/rnl bovine thrombin (T4648, Sigma), 5 mM Ca
  • Rectus femoris muscle tissue was collected at the end of day 10 time point. Injured and contralateral healthy hind limbs were removed at the pubic symphysis and skinned. Both limbs were then placed in 40 ml of 10% neutral buffered formalin for 48 h at room temperature to fix the soft tissue. Following fixation, the defect area was isolated by removing the hip joint and tibia; preserving the quadricep muscles spanning from the patella across the length of the femur. The legs were whole mounted, and paraffin sectioned (4 pm) longitudinally across the middle of the patella indicating midpoint of defect. Sections were stained with standard histological procedures for Mason’s Trichrome and imaged using the Arperio slide scanner (Leica Biosystems, USA). MicroCT
  • Muscle regeneration was evaluated 10 days post-treatment by microCT as a percentage of muscle volume against contralateral healthy legs. Briefly, both hind legs were scanned with microCT with the Nucline NanoScan 3.04.020.0000 (RS 2 D) operated to retrieve 480 projections operating at energy of 50 kVp/670 pA with exposure time set to 300 ms. Scans were performed at maximum zoom with a zigzag motion. Reconstructed scans in 3D were analysed with a licensed PMOD imaging software v4.4 (PMOD Technologies LLC). Quantification of muscle volume was performed by creating a 7mm x 7mm x 7mm ROI aligned along the length of the femur and at the edge of the patella. A threshold of -350 to 800 was applied and volume was measured within the designated ROI. Volume of muscle defect was calculated over volume of healthy contralateral muscle to generate a percentage of muscle volume restored.
  • RS 2 D Nucline NanoScan 3.04.020.0000
  • Normal human skeletal muscle satellite cells were purchased from Lifeline Cell Technology. Cells were thawed in a 37°C waterbath and plated in pre-warmed StemLifeTM Sk complete medium supplemented with antimicrobial (Gentamicin 30mg/ml, Amphotericin B 15 pg/ml at a density of 5,000 cells per cm 2 . Seeded cells were incubated at 37°C, 5% CO2 for 24 h and refed to remove cryopreservation reagents. Cells reaching 70% confluency and actively proliferating were passaged or used for the following proliferation assay. To passage cell, aspirate medium and rinse with PBS.
  • TrypLETM Express Enzyme (12604021 , GibcoTM) was added into the culture vessel and incubated at 37°C, 5% CO2 for 3 min to release the cells. Cells were detached by gently agitation and Trypsin was neutralised with equal volumes of PBS, collected by centrifugation at 150 x g for 5 min for passaging or in vitro proliferation assay.
  • HAVECs Human umbilical vein endothelial cells pooled were purchased from Cell Applications, Inc (200P-05N, Sigma-Aldrich). Cells were thawed in a 37°C waterbath and plated in pre-warmed endothelial cell growth medium (211-500, Sigma- Aldrich). Seeded cells were incubated at 37°C, 5% CO2 for 24 h and refed to remove cryopreservation reagents. Media was refed every other day until cells reach 60% confluency and passaged when cell density reaches 80% confluency. To passage cell, aspirate medium and rinse with PBS.
  • TrypLETM Express Enzyme (12604021, GibcoTM) was added into the culture vessel and incubated for 1 minute at room temperature to release the cells. Cells were detached by gently agitation and Trypsin was neutralised with equal volumes of PBS, collected by centrifugation at 220 x g for 5 min for passaging or in vitro proliferation assay.
  • Human IgGIFc NAMPTcif422-49i fusion protein was designed according to the structure shown in Figure 1.
  • IgGIFc NAMPT422-491 gene was cloned into GeneArt expression ready vector pcDNATM3.4 TOPOTM, a constitutive mammalian expression vector which provides high yield transfections.
  • the vector is 6994 bps long and consists of a CMV promoter, a Woodchuck post-translational regulatory expression (WPRE) downstream to the cloning site to enhance transcript expression and a plIC origin to ensure high copy plasmid replication.
  • WPRE Woodchuck post-translational regulatory expression
  • the IgGIFc NAMPT422-491 gene is 984 bps long spanning from nucleotide 742 to 1725 and flanked by XBal restriction site at the 5’ end and an Agel restriction site at the 3’ end of the design construct.
  • the recombinant pcDNATM3.4 TOPOTM-lgG1FcNAMPT422-49i was transformed into E.Coli DH5a competent cells (EC0111, Thermo Scientific) according to manufacturer’s instructions for amplification.
  • the transfected bacteria were amplified in LB-media with 100pg/ml ampicillin (A0166-5G, Sigma-Aldrich) overnight at 37°C with 225 rpm agitation. Vectors were isolated from transformants by PurelinkTM HiPure Plasmid Maxiprep Kit (K210007, Thermo Scientific) and was verified by restriction enzyme mapping.
  • Expi293 expression system (A14635, Thermo Fisher) was used to express the recombinant proteins. Cells were maintained according to manufacturer’s instructions as described below. Briefly, Expi293 cells were cultured in Expi293 medium (A1435101, ThermoFisher) at 37°C/8%CO2 with 125 rpm agitation with 80% humidity in ventilated capped flasks (Corning® Erlenmeyer sterile polycarbonate with 0.2 pm ventilated caps). The CO 2 Resistant Shakers and Packages (88881104 ThermoFisher) was used for the incubation and these conditions were maintained throughout the culture. For maintenance and subculturing, cells were seeded at a density of 0.4x10 6 viable cells/ml for 3 days until they reached a density of 3-5x10 6 /ml.
  • Expi 293 cells of at least 95% viability and had not reached a density of more than 3-5x10 6 /ml was used.
  • Opti-MEM medium 31985062, ThermoScientific
  • NAMPT422-491 with and IgGIFc domain can be readily purified with a HiTrap Protein A HP 5 ml column (GE17-0403-01, Cytiva).
  • the filtered supernatant was loaded into the HiTrap Protein A column attached to an AKTA START SYSTEM (29022094, Cytiva).
  • the proteins were bound according to protein A column protocol with 20mM sodium phosphate, pH 7.0 and eluted with 0.1M citric acid, pH 4.0. The elution was monitored by measuring the absorbance at 280nm.
  • the buffer was exchanged twice by dialysis in a Slide-A-Lyzer G2 dialysis cassette with a 10K MW cut-off (87731, ThermoFisher).
  • the proteins were concentrated with Amicon ultra centrifugal filters (UFC900308, Merck) with a 10 kDa MW cut-off.
  • the concentrated protein was then filtered through a 0.22 micron syringe filter. Protein concentration was measured with a standard Pierce BCA Protein assay kit (23235, ThermoFisher) and corrected with extinction coefficient calculated from the Expasy protein parameter software.
  • the purified proteins were stored in low-bind tubes at - 80°C.
  • HUVEC cells at 70% confluency were pl replated at 2,000 cells/ well in a 96 well-flat bottom plate in pre-warmed MCDB 131 medium supplemented (10372019, GIBCO). Cells were then treated with 20 nM of equimolar proteins and cultured for 48 h at 37°C/5% CO2. At the end of the timepoint, the microplate was gently inverted and blotted onto paper towels to remove medium from the wells and frozen at -80°C. For quantification, the plates were thawed and room temperature and cell proliferation were measured using the CyQUANTTM Cell Proliferation Assay Kit (C7026, ThermoFisher). Fluoresence was measured on a fluorescence micro-plate reader at 480 nm.
  • Larval zebrafish muscle injury and EdU pulse-chase [0294] Zebrafish larvae (4 dpf) were anaesthetized in 0.01% tricaine (MS-222) (Sigma-Aldrich) in Ringer’s solution. Needle-stab injury was carried out in the dorsal myotome, consisting in a single 30-gauge needle puncture that generates an extensive injury with many damaged muscle fibres. NAMPT and lgG1-NAMPT treatments were carried out by incubating 4 dpf needle-stab-injured larvae in 57 nM of each treatment in Ringer’s solution immediately after injury.
  • Needle-stab-injured larvae at 6 dpf (2 dpi) were transferred into Ringer’s solution containing 50 pg ml-1 Edll (Thermo Fisher Scientific) for 1 h and chased for a further 1.5 h before fixation.
  • Samples were developed using the Click-iT Edll Alexa Fluor 647 imaging Kit (Thermo Fisher Scientific) following the manufacturer’s protocol, followed by a phalloidin immunostaining (Thermo Fisher Scientific).
  • EdU+ cells in a region encompassing two myotomes on either side of the injury were quantified as the number of EdU+ cells outside the injury region.
  • EdU+ cells in the caudal haematopoietic tissue were excluded from this analysis.
  • Statistical analysis has been conducted using Two-way ANOVA with Tuckey’s multiple comparison test.
  • Example 2 Human NAMPT variants stimulate human satellite cell proliferation
  • the inventors sought to compare the activity of lgG1-NAMPT422-49i on human satellite cell proliferation. Human primary satellite cells were treated with 20nM full lgG1-NAMPT422-49i for 48 hours.
  • Example 4 Human NAMPT variants stimulate human endothelial cell proliferation
  • lgG1-NAMPT422-49i displayed increased endothelial cell proliferation compared to PBS-negative control (Fig. 3).
  • Example 5 Minimal versions of NAMPT protein enhance proliferation in response to muscle injury in zebrafish larvae
  • the inventors tested the ability of lgG1-NAMPT422-49i to stimulate cell proliferation in an in vivo zebrafish muscle injury response model.
  • Treatment with lgG1- NAMPT422-491 following needle-stick muscle injury to the zebrafish larvae induced a significant increase in cell proliferation within the injury zone compared to BSA negative or IgG 1 alone controls (Fig. 4A-B & Fig. 5A-B).
  • lgG1-NAMPT422-49i did not stimulate cell proliferation external to the injury zone.
  • volumetric muscle loss is an injury paradigm usually refractory to endogenous-stem cell mediated repair processes and is an area of unmet-clinical need.
  • lgG1-NAMPT422-49i could accelerate regeneration in a mouse model of volumetric muscle loss. Strikingly, delivery of lgG1-NAMPT422-49i into the muscle defect via a fibrin hydrogel, but not a fibrin only control hydrogel, was able to fully restore muscle architecture when applied to the wound site (Fig. 6A-B).
  • the increase in muscle volume in mice treated with lgG1-NAMPT422-49i was also greater than all other treatment groups, including NAMPT422-491 without an lgG1 fusion.
  • Treatment groups [0304] Groups of 5-6 weeks of age C57BI/6J male and female mice were treated on Study day 1 with lgG1-NAMPT422-49i by the intravenous route as either a single bolus dose of 1 , 3, 7.5 and 10 pg/mouse (“single dose study”) or as a daily bolus dose at dose levels of 1, 3 and 10 pg/mouse over a period of seven days (“repeated dose study”).
  • the repeated dose study included a vehicle control group of 0.9% saline.
  • the treatment groups for the single dose phase and repeated dose phase are provided in Tables 4 and 5 below.
  • the clinical observations included examination of animals for changes in skin and fur, eyes and mucous membranes, respiratory and circulatory function, gait and posture, behaviour, tremors or convulsion and any other abnormal findings. This also included daily examination of the tail for injection site reactions. Any other injection site lesions such as eschar, necrosis and ulceration were also noted.
  • mice were fasted for at least 4 h prior to terminal bleed sampling with access to food withdrawn but water available. After administration of an overdose of pentobarbitone to anaesthetise the animal, blood sample were collected puncture of the inferior vena cava.

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Abstract

La présente invention concerne la réparation et la régénération des tissus productifs, et plus particulièrement les protéines de fusion, les compositions comprenant lesdites protéines de fusion, et les procédés d'utilisation desdites protéines de fusion ou compositions pour la réparation et la régénération des tissus productifs. Selon un aspect, l'invention concerne une protéine de fusion comprenant : un polypeptide NAMPT à doigt de cytokine (cif) et une région Fc d'un anticorps.
PCT/AU2024/050432 2023-05-03 2024-05-03 Protéines de fusion à base de nampt Ceased WO2024227231A1 (fr)

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